One of the Nine Doublet Microtubules of Eukaryotic Flagella Exhibits Unique and Partially Conserved Structures

Abstract

The axonemal core of motile cilia and flagella consists of nine doublet microtubules surrounding two central single microtubules. Attached to the doublets are thousands of dynein motors that produce sliding between neighboring doublets, which in turn causes flagellar bending. Although many structural features of the axoneme have been described, structures that are unique to specific doublets remain largely uncharacterized. These doublet-specific structures introduce asymmetry into the axoneme and are likely important for the spatial control of local microtubule sliding. Here, we used cryo-electron tomography and doublet-specific averaging to determine the 3D structures of individual doublets in the flagella of two evolutionarily distant organisms, the protist Chlamydomonas and the sea urchin Strongylocentrotus. We demonstrate that, in both organisms, one of the nine doublets exhibits unique structural features. Some of these features are highly conserved, such as the inter-doublet link i-SUB5-6, which connects this doublet to its neighbor with a periodicity of 96 nm. We also show that the previously described inter-doublet links attached to this doublet, the o-SUB5-6 in Strongylocentrotus and the proximal 1–2 bridge in Chlamydomonas, are likely not homologous features. The presence of inter-doublet links and reduction of dynein arms indicate that inter-doublet sliding of this unique doublet against its neighbor is limited, providing a rigid plane perpendicular to the flagellar bending plane. These doublet-specific features and the non-sliding nature of these connected doublets suggest a structural basis for the asymmetric distribution of dynein activity and inter-doublet sliding, resulting in quasi-planar waveforms typical of 9+2 cilia and flagella.

The nexin link and B‐tubule glutamylation maintain the alignment of outer doublets in the ciliary axoneme

“…Some of the tomograms were previously used for the analysis of other axonemal complexes (Heuser et al 2012a; Heuser et al 2009; Lin et al 2012). Subtomogram averaging of the 96-nm axonemal repeats was performed using the Particle Estimation for Electron Tomography software (Nicastro et al, 2006) available from the Boulder Laboratory for 3D Electron Microscopy of Cell (Colorado).…”

The Neomuran Revolution and Phagotrophic Origin of Eukaryotes and Cilia in the Light of Intracellular Coevolution and a Revised Tree of Life

“…Protocentrioles were rigidified by a hubspoke core, microtubules fixed at nine by ninefold SAS-6 hub assembly (Guichard et al 2012). Cross-links rigidified the axoneme, the two on its substratum face having numerous extra-rigid linkers linking them like sled runners (subsequently becoming doublets 1 and 2 of Chlamydomonas: Lin et al 2012). Novel proteins (e.g., Rib43a: Norrander et al 2000) stabilized A-tubules and the bases of centriolar root microtubules (dorsal and ventral fans) compared with transient spindle microtubules.…”

Turning dyneins off bends cilia

“…Importantly, in sea urchin sperm axonemes a bridging structure (termed o‐SUB5–6), rather than dynein arms, connects doublet microtubules 5 and 6 (Lin et al, ). The bridge is thought to form a rigid component within the nine outer doublets that runs essentially parallel to the plane of the central pair microtubule complex whose orientation in sea urchin sperm is also enforced by inelastic linkers.…”